Botanical Journal of the Linnean Society, 2009, 160, 211–231. With 6 figures Phylogenetic significance of leaf micromorphology and anatomy in the tribe Mentheae (Nepetoideae: Lamiaceae) boj_979 211..231 HYE-KYOUNG MOON1*, SUK-PYO HONG SUZY HUYSMANS1 FLS2, ERIK SMETS FLS1,3 and 1 Laboratory of Plant Systematics, Institute of Botany and Microbiology, K.U. Leuven, Kasteelpark Arenberg 31, PO Box 2437, BE-3001 Leuven, Belgium 2 Laboratory of Plant Systematics, Department of Biology and Institute of Global Environment, Kyung Hee University, Seoul 130-701, South Korea 3 National Herbarium of the Netherlands, Leiden University Branch, PO Box 9514, NL-2300 RA Leiden, The Netherlands Received 25 December 2008; accepted for publication 1 April 2009 A comparative micromorphological study of leaves was carried out on 102 species of Mentheae; 61 species were selected for the anatomical study. Mentheae possessed both amphistomatic and hypostomatic leaves. The diversity of leaf epidermal characteristics was based on the variation in morphology of epidermal cells, stomata types and trichome types. Although each characteristic on its own has rather limited systematic value, the combination of some of these features may be systematically relevant, especially for the identification of species. For example, branched multicellular nonglandular trichomes were a diagnostic characteristic for all genera investigated of the subtribe Salviinae; however, this trichome type was also observed in Hedeoma ciliolata and Neoeplingia leucophylloides of the subtribe Menthinae. Capitate glandular trichomes with pear-shaped heads were only observed in Salvia dorrii. Subsessile glandular trichomes with multicellular heads (more than ten cells) were an apomorphy for Perovskia. The anatomical leaf structure was consistent throughout the tribe. In some species, the vascular bundles in the midrib were modified into a mechanical tissue, which is an adaptation to xerophytic environments. The observed variations are discussed in an ecological context and their phylogenetic significance is evaluated. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231. ADDITIONAL KEYWORDS: epidermis – phylogenetics – stomata – systematics – trichome – vascular bundle. INTRODUCTION Lamiaceae (Lamiales, euasterids I; APG II) are rich in herbs and medicinal plant species, which are of great economic importance. Lamiales formerly had a restricted circumscription that included the families Lamiaceae, Verbenaceae, Boraginaceae and Lennoaceae (Cronquist, 1988), but recent phylogenetic work has shown that Lamiales is monophyletic with the inclusion of former orders Bignoniales, Hippuridales, Plantaginales and Scrophulariales (Stevens, *Corresponding author. E-mail: hyekyoung.moon@bio.kuleuven.be 2001; Judd et al., 2008). Consequently, Lamiales has become one of the larger angiosperm groups, containing about 12% of the eudicot diversity (23 families with 1059 genera and 23 275 species; Stevens, 2001). Lamiaceae has a cosmopolitan distribution and consists of 236 genera and about 7000 species (Stevens, 2001). Many species of Lamiaceae produce essential oils which are secreted by glandular hairs on aerial vegetative organs and some reproductive organs. These hairs have been investigated from structural, ultrastructural and biochemical viewpoints for their commercial value in various members of the family (Amelunxen, 1964, 1965; Amelunxen, Wahlig & © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 211 212 H.-K. MOON ET AL. Arbeiter, 1969; Heinrich, 1973; Bosabalidis & Tsekos, 1982, 1984; Heinrich et al., 1983; Werker, Putievsky & Raavid, 1985; Dudai et al., 1988; Bosabalidis, 1990; Autunes & Sevinate-Pinto, 1991). Although leaf epidermal morphology has received less attention than fruit morphology and seed anatomy as a potential systematically informative characteristic, the surface of the leaves is commonly covered by various nonglandular and glandular trichomes which, with characteristics of the stomatal complex, may have systematic value (Stace, 1984; Webster, Del-ArcoAguilar & Smith, 1996; Moon & Hong, 2003; Beilstein, Al-Shehbaz & Kellogg, 2006). In Lamiaceae, the systematic value of trichome types was demonstrated by Abu-Asab & Cantino (1987) in the subtribe Melittidinae (Dumort.) Endlicher. In addition, Cantino (1990) performed a comprehensive study of Lamiaceae and Verbenaceae with emphasis on the morphology of the stomatal complex and the subsessile glandular trichomes, including their systematic importance. However, this study focused mainly on the subfamily Lamioideae sensu Erdtman (1945) in order to elucidate the relationships with Verbenaceae, rather than subfamily Nepetoideae (Dumort.) Luerssen. Since Erdtman (1945) suggested two subfamilies in Lamiaceae (Lamioideae and Nepetoideae) on the basis of pollen characteristics, the monophyly of subfamily Lamioideae has been questioned because of similarity with Verbenaceae (Cantino & Sanders, 1986). Consequently, in the most recent classification of Lamiaceae (Harley et al., 2004), Lamioideae sensu (Erdtman, 1945) is segregated into six subfamilies, Symphorematoideae Briq., Viticoideae Briq., Ajugoideae Kostel., Prostantheroideae Luerss., Scutellarioideae (Dumort.) Caruel and Lamioideae Harley, which include many genera of former Verbenaceae. Nepetoideae is always supported as a monophyletic group in both molecular and morphological analyses (Cantino & Sanders, 1986; Cantino, Harley & Wagstaff, 1992; Kaufmann & Wink, 1994; Wagstaff, Olmstead & Cantino, 1995; Harley et al., 2004). A comparative anatomical study of leaves has been reported only for subtribe Hyptidinae Endl. of Nepetoideae (Rudall, 1980). Rudall (1979) also showed that the variation in leaf structure is correlated with ecological constraints, but anatomical data are useful for infrageneric delimitation in Nepetoideae (Hyptidinae: Rudall, 1979, 1980; Origanum L.: Bosabalidis & Kokkini, 1997). Mentheae Dumort. is the largest tribe of Nepetoideae, including 65 genera and approximately 2000 species, and these species are known for their high content of essential oils, which are widely used in pharmaceutical preparations, perfumery and cosmetics. According to the most recent classification, Mentheae can be divided into three subtribes: Salviinae (Dumort.) Endl., Nepetinae (Dumort.) Coss. & Germ. and Menthinae (Dumort.) Endl. (Harley et al., 2004). Although Mentheae is a well-supported monophyletic group within Nepetoideae, the relationships among the genera are poorly resolved. In particular, the relationship of Heterolamium C.Y.Wu and Melissa L. to other members of Mentheae remains obscure (Wagstaff et al., 1995; Harley et al., 2004). The lack of morphological support for the molecular phylogenetic hypothesis of Mentheae illustrates the necessity of thorough morphological research. Our previous research has focused on the systematic relationships of Mentheae sensu Harley et al. (2004) based on pollen and nutlet characteristics. These features proved to be systematically important at various taxonomic levels, and especially so at the generic level (Moon, 2008; Moon et al., 2008a, 2008b). These results encouraged us to explore other morphological characteristics, such as leaf features, in order to further our understanding of the phylogenetic relationships within the study group. The objective of this paper is therefore to present a detailed description of leaf micromorphology in Mentheae using scanning electron microscopy (SEM). In addition, we aim to describe the leaf anatomy based on the midrib structure of selected taxa. The variation in leaf characteristics is discussed with respect to their potential systematic value and in relation to our previous work in Mentheae. MATERIAL AND METHODS This study was conducted mainly on material taken from herbarium specimens on loan from the following herbaria (abbreviations according to Holmgren, Holmgren & Barnett, 1990): BR, G, GH, K, LV, MO, S and SNU. Plants recently collected in formalin–acetic acid–alcohol (FAA) by H.-K. Moon were also used. A list of all species and specimens investigated is provided in the Appendix. This study included all genera of Mentheae sensu Harley et al. (2004), except for the monotypic Eriothymus J.A.Schmidt, which is known only from the type collection and is possibly extinct (Harley et al., 2004). Lycopus L. was studied only anatomically in this study because the detailed leaf morphology was carried out by Moon & Hong (2003). For leaf epidermal observations, leaves were first examined using a stereomicroscope (Leica MZ6) in order to select fully mature leaves. The dried material was rehydrated overnight in the wetting agent Agepon® (Agfa Gevaert, Leverkusen, Germany; Agepon wetting agent : distilled water, 1 : 200) prior to dehydration. Leaves were separated from the specimen/individual and the mid-part of the leaf was dissected with a razor blade to fit the size of the aluminium stub. All samples were dehydrated © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE through a graded ethanol series (in 70, 90, 95 and 100% ethanol) prior to critical point drying (CPD 030, Balzers). The dried leaves were mounted on stubs with double adhesive tape. The stubs were coated with gold (SPI-MODULE™ Sputter Coater, SPI Supplies, West Chester, PA, USA) and observed with a JEOL JSM-6360 scanning electron microscope at 5–15 kV. For the anatomical observations, the material was dehydrated in a graded ethanol series, embedded in paraffin, sectioned (8–10 mm) with a rotary microtome (Zeiss HM360), stained in aniline blue and safranin solutions and permanently mounted using Entelan. Sections were examined and photographed using a Leitz Dialux 20 light microscope equipped with a PL-B622CF microscopy camera (PixeLink) and Microscopica v1.3 (Orbicule, Belgium). Size measurements on SEM and light microscopy images were made using Carnoy 2.0 (Schols et al., 2002). Terminology for the stomatal complex, trichomes and surface sculpturing follows Cantino (1990) and Moon & Hong (2003). RESULTS Significant variation was observed in epidermal cells, the stomatal complex and trichomes (Figs 1–4). The midrib structure of the leaf was similar throughout the tribe (Fig. 5), but xeromorphic adaptations were found in a few species (Fig. 5H, I). Representative leaf characteristics are summarized in Table 1. In some cases, the stomatal complex and cell surface pattern could not be recognized because of extremely dense nonglandular trichomes on the epidermis. EPIDERMAL CELLS Leaf epidermal cells were irregular or isodiametric (Table 1). The cell outlines were described for adaxial (AD) and abaxial (AB) leaf surfaces separately, although in many taxa the patterns were similar on the same leaf. Irregular cells were rather common and distributed evenly on both sides. Isodiametric cells were more common on AD surfaces (c. 62%). Undulate cell outlines were found in Bystropogon canariensis (L.) L’Hér (AD), Dicerandra odoratissima R.M.Harper (AB), Hoehnea minima (Schmidt) Epling (AB), Meehania urticifolia (Miq.) Makino (AD), Salvia polystachya Ort. (AB) and Salvia sclarea L. (AB and AD). Sometimes, the epidermal cells were invisible because of a dense layer of trichomes (Figs 1D, E, I, 2E). The isodiametric epidermal cells usually had straight to curved anticlinal walls, whereas irregular cells had undulate to sinuate anticlinal walls. The anticlinal walls of undulate cell outlines were hardly discernible (Figs 1, 2). 213 Striation occurred commonly on irregular cells and in some taxa on isodiametric cells (Table 1). Striation often occurred on only one side of the leaf (AB, 14 species; AD, eight species), except in 16 species where striation was observed on both sides of the leaf. Striae were restricted to the stomatal areas in Hesperozygis nitida (Benth.) Epling (AB), Micromeria marginata (Sm.) Chater (AD; Fig. 2J, K), Poliomintha glabrescens A.Gray ex Hemsl. (AD) and Satureja thymbra A.Gray ex Hemsl. (AD; Fig. 2O). STOMATA Representatives of Mentheae had both amphistomatic and hypostomatic leaves, but amphistomatic (69 species) were more common than hypostomatic (25 species; Table 1) leaves. Meriandra bengalensis (Roxb.) Benth. may be amphistomatic with a few stomata on the AD surface, although stomata were invisible on the AB surface. Bystropogon canariensis, Conradina canescens A.Gray, Conradina grandiflora Small, Hedeoma ciliolata (Epling & Stewart) Irving, Poliomintha longiflora A.Gray, Rosmarinus officinalis L. and Salvia dorrii (Kellogg) Abrams are possibly hypostomatic as stomata were absent on the AD side. In Mentheae, five types of stomatal complex were observed (definitions adapted from Cantino, 1990; and references cited therein): actinocytic (stoma surrounded by a single ring of five or more radially elongated cells enclosing the guard cells); anisocytic (stoma surrounded by three subsidiary cells, one of which is markedly smaller than the other two); anomocytic (stoma surrounded by cells that are indistinguishable from other epidermal cells); diacytic (stoma enclosed by a pair of subsidiary cells with common walls perpendicular to the guard cells); diallelocytic (stoma enclosed by an alternating complex of three subsidiary cells of graded size oriented perpendicular to the guard cells). In some cases, more than one type of stoma may be present on the same surface, for example, anomocytic, anisocytic and diacytic in Glechoma hederacea (Fig. 1P). Moreover, diacytic and diallelocytic types often occurred simultaneously (Table 1; Fig. 2G, M, O). The most common types of stomata in the tribe were diacytic and anomocytic. In the amphistomatic taxa, stomata were more frequent on the AB surface. Stomatal size varied considerably in Mentheae (10–36 ¥ 7–27 mm). The smallest stoma was recorded in Zataria multiflora Boiss. on the AD surface (10 mm in length), and Thymus serpyllum L. possessed the largest stoma on the AB surface (36 mm in length). TRICHOMES The leaf epidermis of all species investigated was covered by various hairs. We defined two categories of © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 214 Table 1. Characterization of the leaf components in Mentheae Subtribe Salviinae Chaunostoma mecistandrum Dorystaechas hastata © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 Lepechinia calycina Lepechinia caulescens Meriandra bengalensis Perovskia abrotanoides (thrum) Perovskia abrotanoides (pin) Perovskia scrophulariifolia Rosmarinus officinalis Salvia aethiopis Salvia canariensis Salvia coccinea Salvia dorrii Salvia glutinosa Salvia officinalis Salvia polystachya Salvia pratensis Salvia rypara Salvia sclarea Salvia taraxacifolia Salvia verbenaca Salvia verticillata Zhumeria majdae Subtribe Menthinae Acanthomintha lanceolata Acanthomintha obovata SP AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB + + + + + + + + ± ++ ++ ++ ++ + + ± + + + + + + + + + + + + + + + + + + + + + + + + + + AD AB AD AB + + + + Stomatal type Size of stomata (mm) / / / Ani/dia/diallelocytic Anomocytic Diacytic Anomocytic # 13–16 ¥ 12–17 22–24 ¥ 18–23 24–28 ¥ 21–23 17–20 ¥ 13–15 18–20 ¥ 16–18 20–25 ¥ 15–16 18–23 ¥ 16–18 15 ¥ 9 Dia/diallelocytic Dia/diallelocytic Dia/diallelocytic / Anomo/diacytic Diacytic 16–19 ¥ 14–16 16–20 ¥ 13–15 19–22 ¥ 15–19 18–22 ¥ 15–17 20–24 ¥ 15–18 16–21 ¥ 15–18 Diacytic / Dia/diallelocytic / / Anomo/actinocytic / / 16–19 ¥ 13–16 13–18 ¥ 13–15 20–24 ¥ 14–17 15–21 ¥ 11–15 19 ¥ 12 19–22 ¥ 16–20 18–22 ¥ 20–22 20–25 ¥ 18–20 / Diacytic # 16–24 ¥ 13–16 12–15 ¥ 12–15 / / Diacytic / Anomocytic / / Anomocytic / Diacytic Diacytic Anomocytic Anomocytic # # 13–17 ¥ 13–15 16–18 ¥ 14–16 20–25 ¥ 14–18 22–27 ¥ 19–20 21–25 ¥ 20–22 17–20 ¥ 13–19 13–17 ¥ 13–14 18–22 ¥ 16–18 19–24 ¥ 16–18 20–25 ¥ 15–19 21–25 ¥ 18–20 12–15 ¥ 9–10 12–16 ¥ 10–14 Diacytic Anomo/dia/diallelocytic Anomocytic Anomo/diacytic 20–26 ¥ 15–20 16–22 ¥ 16–20 17–20 ¥ 10–13 14–20 ¥ 12–14 Surface cell AW SS SL US UL B C P S Isometric / Striated Striated isometric Isometric Irregular Irregular Irregular # # Irregular Irregular Irregular Striated irregular Irregular Irregular Isometric Irregular Irregular / Isometric / / Partially striated irregular Partially striated / Irregular Irregular Isometric # Irregular / Isometric Irregular / Isometric / / Irregular Irregular Isometric Irregular Partially striated irregular Partially striated irregular # # st/cur / / st/cur cur/un sin cur un + + + + + + + + + + + + + + + + + + + + + + - ++ ++ + + - + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + vf + + + + + - + + + + + + + + ++ ++ ++ ++ + + + ++ ++ + + + + + + + + + + + + + vf + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + ++ ++ ++ ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + - - + + + - - + + + + + + + + + Irregular Irregular Irregular Irregular un un sin / st/cur un cur st un / cur/un / / sin / / sin sin st/cur sin / cur/un un/sin / st/cur / / un un st/cur st/cur/un sin sin st/cur cur sin sin H.-K. MOON ET AL. Taxon Bystropogon canariensis Cleonia lusitanica Clinopodium acinos Clinopodium chinense Clinopodium vulgare Conradina canescens Conradina grandiflora Cuminia eriantha var. fernandezia Cunila origanoides Cyclotrichium origanifolium Dicerandra christmanii Dicerandra odoratissima Glechon marifolia Gontscharovia popovii Hedeoma ciliolata Hesperozygis nitida Hoehnea epilobioides Hoehnea minima Horminum pyrenaicum Hyssopus officinalis Kurzamra pulchella Mentha pulegium Micromeria marginata Minthostachys andina Minthostachys mollis Monarda fistulosa Monarda punctata + ± + ++ + + + + ± ± + + + + + + + + + + + + ± ++ + + + + + + + + + + + + ++ + + + + + + + + Anomocytic 22–25 ¥ 13–19 Anomocytic Anomo/diacytic Anomocytic Anomo/diacytic 23–29 ¥ 17–20 22–29 ¥ 17–20 16–19 ¥ 11–15 13–19 ¥ 12–16 Anomocytic 12–14 ¥ 8–10 Anomo/diacytic 13–19 ¥ 11–15 Anomocytic 12–16 ¥ 7–10 Diacytic Diacytic Diacytic Anomocytic Anomo/diacytic Diacytic Diacytic Diacytic Diacytic / / 15–18 ¥ 10–13 16–19 ¥ 14–16 13–21 ¥ 13–18 19–24 ¥ 14–17 18–25 ¥ 15–19 18–23 ¥ 13–16 15–22 ¥ 11–14 30–33 ¥ 20–22 24–29 ¥ 18–21 17–22 ¥ 11–17 16–21 ¥ 14–17 Striated actinocytic 16–21 ¥ 12–17 Actinocytic / Anomo-diacytic Dia/diallelocytic Diacytic Diacytic Diacytic Dia/diallelocytic Dia/diallelocytic Dia/diallelocytic Dia/diallelocytic Striated anomo/actinocytic Actinocytic 19–23 ¥ 14–16 14 ¥ 8 13–17 ¥ 10–13 17–20 ¥ 12–14 16–19 ¥ 12–14 25–31 ¥ 19–22 26–32 ¥ 19–22 20–24 ¥ 17–20 17–22 ¥ 13–17 20–24 ¥ 18–20 18–20 ¥ 15–17 18–25 ¥ 13–16 17–19 ¥ 13–15 Anomocytic 19–22 ¥ 15–19 Anomocytic / / / / Diacytic Diacytic 17–25 ¥ 14–16 18–21 ¥ 14–17 14–17 ¥ 13–15 17–19 ¥ 13–15 15–21 ¥ 12–14 31–34 ¥ 23–25 27–33 ¥ 22–27 Irregular Irregular / # Striated irregular Striated irregular Irregular Irregular Irregular Irregular Irregular Irregular / # Isometric # Striated isometric Striated isometric Irregular Irregular Irregular Irregular Striated isometric Striated irregular Irregular / Irregular Irregular / / Irregular Irregular Irregular Irregular Isometric Partially striated irregular / / Isometric Striated irregular Isometric Partially striated irregular Partially striated irregular Partially striated irregular Irregular Irregular Irregular Striated irregular Irregular Striated irregular Isometric Irregular Irregular / Irregular Irregular Partially striated irregular Partially striated irregular sin sin / sin sin un cur/un un/sin sin sin sin / st/cur cur cur sin un sin sin cur cur/un cur / sin un / / un un st/cur un/sin st/cur cur / / st/cur un st/cur cur/un sin sin sin sin sin sin sin cur/sin st/cur sin cur/un / un sin cur/un cur/un + + ++ + + + + + + vf + vf + + + ++ + + + + + + + + + + + + + + vo + + + + + + + + + + + + + + + + + + - + + ++ ++ + + + + + + vo + + + + + + + + + + + + ++ + + + + + vf + + + + + + + + + + + mo - + ++ - + + + + + + + + + + vf + + vf + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 215 Monardella macrantha AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 Blephilia ciliata 216 Table 1. Continued Monardella nana Monardella odoratissima © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 Neoplingia leucophylloides Obtegomeria caernlescens Origanum rotundifolium Origanum vulgare Pentapleura subulifera Piloblephis rigida Pogogyne douglasii Pogogyne serpylloides Poliomintha glabrescens Poliomintha incana Poliomintha longiflora Prunella vulgaris Pycnanthemum albescnes Pycnanthemum incanum Rhabdocaulon coccineum Rhabdocaulon strictum Rhododon ciliatus Saccocalyx satureioides Satureja thymbra Stachydeoma graveolens Thymbra spicata Thymus pallasianus Thymus serpyllum Zataria multiflora AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB SP Stomatal type Size of stomata (mm) + ++ + + + + + + + + + + ± + + + + + + + ± + + + + + + + + + + + + + + + + + + + + + + Dia/diallelocytic Dia/diallelocytic Anomocytic Anomocytic 27–31 ¥ 22–24 28–37 ¥ 19–22 13–14 ¥ 11–13 15–18 ¥ 13–17 / 13 ¥ 14 / Aniso/dia/diallelocytic Dia/diallelocytic Anomocytic Anomo/dia/diallelocytic Diacytic Diacytic / 20–24 ¥ 15–20 17–23 ¥ 13–18 12–15 ¥ 10–12 18–21 ¥ 12–16 17–23 ¥ 12–16 12–18 ¥ 12–16 Diacytic Diacytic Anomocytic Anomocytic Striated diacytic Anomocytic 24–27 ¥ 16–20 21–24 ¥ 16–18 19–22 ¥ 14–17 13–16 ¥ 12–14 24–28 ¥ 16–19 21–26 ¥ 16–19 / 14–28 ¥ 14–17 Dia/diallelocytic Dia/diallelocytic 27–30 ¥ 19–22 20–25 ¥ 17–20 / 18–21 ¥ 14–18 Anomocytic 19–24 ¥ 18–20 Dia/diallelocytic 22–27 ¥ 15–20 Diallelocytic Anomocytic Anomocytic Diacytic Diacytic Striated diallelocytic Dia/diallelocytic / Diacytic / Diacytic/ / / Diacytic / Dia/diallelocytic Dia/diallelocytic 23–29 ¥ 18–21 22–25 ¥ 17–21 18–21 ¥ 13–18 17–24 ¥ 13–15 15–19 ¥ 12–15 25–28 ¥ 18–20 21–25 ¥ 17–19 16–20 ¥ 15–18 16–19 ¥ 14–17 23–28 ¥ 20–24 24–27 ¥ 17–22 21–23 ¥ 15–17 20–23 ¥ 13–16 26–30 ¥ 21–23 30–36 ¥ 18–20 10–13 ¥ 8–12 15–18 ¥ 10–14 Surface cell AW SS SL US UL B C P S Isometric Partially striated irregular Irregular / Irregular Irregular Striated Striated isometric Isometric Isometric Isometric Irregular Partially striated irregular Irregular Isometric Striated isometric Partially striated isometric Partially striated irregular Partially striated irregular Partially striated irregular Irregular / Striated Striated Irregular # Striated isometric / # # Irregular / Isometric Irregular Striated irregular Isometric Partially striated irregular Partially striated irregular / / Irregular Irregular Irregular / Partially striated isometric Isometric Striated irregular Striated irregular Irregular Striated irregular Irregular / st/cur cur/un cur / st/cur st/un / cur st/cur st/un cur/un un/sin un cur st/cur st/cur cur un/sin sin sin un / / / sin / un / / / un / st/cur un st/cur st/cur sin sin / / un un un/sin + + + + + + + + mo + m-vf ++ + + + + + + mo + + + + + + + vo + mf + + + - + + + + + + mf + mo + + ++ + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + m-vf - ++ ++ - + + + ++ + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + cur/un cur/un cur/un cur un cur/un sin / H.-K. MOON ET AL. Taxon Ziziphora clinopodioides Subtribe Nepetinae Agastache urticifolia Cedronella canariensis Dracocephalum parviflorum Dracocephalum ruyschiana Drepanocaryum sewerzowii Glechoma hederacea (hermaphrodite) Glechoma hederacea (female) Hymenocrater bituminosus Lallemantia peltata Lallemantia royleana Lophanthus tschimganicus Marmoritis rotundifolia Meeehania urticifolia Nepeta cataria Nepeta nuda Nepeta fissa Nepeta grandiflora Schizonepeta multifida Schizonepeta tenuifolia Incertae Sedis Heterolamium debile Melissa flava Melissa officinalis AD AB AD AB + + + + Anomo/anisocytic / Anomocytic Anomocytic 15–21 ¥ 12–17 19–24 ¥ 16–20 15–19 ¥ 12–15 16–19 ¥ 12–15 Polygonal with striae / Irregular / / / sin / + + + - + + - + - - + - - + + + AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB AD AB + ++ + ++ + ++ + + + + + + + ++ + ++ + ++ + + + + + + + + + + ++ + + + + Anomocytic Anomocytic 13–17 ¥ 16–20 14–20 ¥ 11–16 13–15 ¥ 10–12 Anomo/diacytic Diacytic Actino/diacytic / Anomo/diacytic 15–20 ¥ 13–17 20–28 ¥ 20–22 24–28 ¥ 19–24 16–21 ¥ 13–17 17–19 ¥ 13–15 Anomo/aniso/diacytic 21–27 ¥ 16–20 Anomo/anisocytic Actino/anomocytic Anomo/diacytic Striated anomo/diacytic Anomo/diacytic Dia/diallelocytic Actinocytic Diacytic Anomocytic / / 21–27 ¥ 16–20 15–18 ¥ 13–17 15–20 ¥ 14–16 13–19 ¥ 14–16 13–19 ¥ 13–16 19–24 ¥ 14–18 19–21 ¥ 18–20 21–24 ¥ 14–17 13–17 ¥ 13–16 13–16 ¥ 11–13 15–20 ¥ 14–19 Anomo/diacytic Anomocytic Anomocytic dia/diallelocytic Anomo/diacytic Diacytic # Diacytic Actino/anomocytic / / Anomocytic Anomo/diacytic 16–20 ¥ 13–15 20–22 ¥ 15–16 15–20 ¥ 13–16 14–18 ¥ 11–13 18–22 ¥ 12–15 18–22 ¥ 14–17 13–17 ¥ 11–13 19–30 ¥ 13–17 19–23 ¥ 14–19 12–15 ¥ 13–18 18–23 ¥ 19–23 18–25 ¥ 15–18 22–26 ¥ 17–21 sin sin sin sin st/cur st/cur st sin sin / sin st sin sin cur/un cur/un sin sin st/cur / sin sin st/cur st / / sin sin sin sin st/cur ++ ++ + Anomocytic Irregular Striated irregular Irregular Striated irregular Isometric Isometric Striated isometric Striated irregular Irregular Striated Partially striated irregular Partially striated irregular Partially striated irregular Irregular Irregular Irregular Irregular Partially striated irregular Isometric / Irregular Irregular Striated isometric Striated isometric / Irregular Irregular Irregular Irregular Irregular Isometric # Irregular Striated irregular Irregular Irregular Irregular Irregular cur/un sin / / / sin + + + + + vf + + + + + ++ + + + vf + - + + + + + + + + + + + + + + + ++ + + - + + + + + + + + + + + + + - + ++ + + + + + + + + + + + + + + + + + + + + + + + + vf + vf + + vf + + + + + - + + + + + + + + + + + + + + + + + + + + AD AB AD AB AD AB + ++ + + / / 13–15 ¥ 13–16 14–16 ¥ 12–14 14–16 ¥ 13–15 / 15–20 ¥ 12–16 sin sin sin sin / sin + + + + - + + + + - + + + + vo + + vo - + + / Irregular Irregular Irregular Irregular Irregular Irregular - + + + + + + + 217 /, undetermined because of irregular shape of guard cells; #, unidentified because of dense trichomes; -, absent; +, present moderately; ++, present abundantly; ±, unknown because of dense trichomes; AB, abaxial side; AD, adaxial side; AW, anticlinal wall shape; B, branched nonglandular trichome; C, capitate glandular trichome; cur, curved; mf, more frequent at leaf margin; mo, observed only at leaf margin; P, pilate glandular trichome; S, subsessile glandular trichome; sin, sinuate; SL, long simple nonglandular trichome; SP, stomatal presence; SS, short simple nonglandular trichome; st, straight; UL, long uniseriate nonglandular trichome; un, undulate; US, short uniseriate nonglandular trichome; vf, more frequent in vein including midrib; vo, observed only in vein including midrib. LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 Ziziphora capitata 218 H.-K. MOON ET AL. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE 219 Figure 1. Scanning electron micrographs of leaf surfaces of subtribes Salviinae and Nepetinae. A, Dorystaechas hastata. Adaxial surface showing cluster of branched nonglandular trichomes. B, Melissa officinalis L. Abaxial surface with simple nonglandular trichomes and anomocytic stomata. C, D, Meriandra bengalensis showing clear venation border with trichome distribution (C, adaxial surface; D, abaxial surface). E, Perovskia abrotanoides. Abaxial surface showing striate irregular epidermal cells. F, Rosmarinus officinalis. Abaxial surface of tightly revolute leaf. G, Salvia officinalis. Adaxial surface. H, Salvia dorrii. Adaxial surface with subsessile glandular trichomes and pear-shaped head attached excentrically to the stalks. I, Agastache urticifolia Kuntze. Adaxial surface showing simple nonglandular trichomes with irregular epidermal cells and sinuate cell walls. J, Cedronella canariensis (L.) Webb & Berthel. Adaxial surface showing irregular epidermal cells with sinuate cell walls. K, L, Dracocephalum ruyschiana L. K, Adaxial surface showing isometric epidermal cells with well-developed striations and simple nonglandular trichomes. L, Abaxial surface showing irregular epidermal cells with striations and actinocytic/diacytic stomata. M, N, Glechoma hederacea. M, Adaxial surface with partially striated irregular epidermal cells with sinuate anticlinal walls. N, Abaxial surface with anomocytic/anisocytic/diacytic stomata. O, Nepeta nuda L. Abaxial surface showing distribution of trichomes on leaf blade and vein. ! these, nonglandular and glandular, according to the absence or presence of a secretory head on the trichome. For the glandular trichomes, we distinguished between capitate and subsessile trichomes following Cantino (1990). Nonglandular trichomes Three types of nonglandular trichome were observed: simple unicellular trichomes, uniseriate trichomes and branched (= dendriform) trichomes (Fig. 3). In the simple unicellular and uniseriate trichomes, we defined two subtypes according to their length. The long simple unicellular trichomes (> 100 mm) were only found in Conradina grandiflora, Salvia officinalis L. and Salvia taraxacifolia Coss. & Bal. on both sides with similar frequency. Long uniseriate trichomes (> 200 mm) occurred throughout Mentheae and were found together with short uniseriate trichomes (Table 1). The simple unicellular and uniseriate trichomes were usually distributed evenly on the leaf, but sometimes occurred more frequently on the margin or vein, or were only found on the margin or vein (Table 1; Fig. 3H). Branched trichomes were found in all genera of Salviinae and also in Hedeoma ciliolata and Neoeplingia leucophylloides Ramamoorthy of Menthinae (Table 1; Fig. 3I–L). Simple unicellular and uniseriate trichome types were common and widespread in Mentheae. Glandular trichomes Three different glandular trichomes were present: capitate trichomes, pilate trichomes and subsessile glandular trichomes (= peltate glandular trichomes; Fig. 4). In capitate glandular trichomes, the head cell was attached to a single cell stalk, whereas, in pilate glandular trichomes, the stalk consisted of more than one cell (Fig. 4A–F). Capitate glandular trichomes occurred in most taxa of Mentheae, but were not found in Conradina canescens, Conradina grandiflora, Dicerandra christmanii Huck & Judd, Drepanocaryum sewerzowii (Regel) Pojark., Gontscharovia popovii (B.Fedtsch. & Gontsch.) Boriss., Hedeoma ciliolata, Lallemantia peltata (L.) Fisch. & C.A.Mey., Marmoritis rotundifolia Benth., Prunella vulgaris L., Schizonepeta multifida (L.) Briq., Zataria multiflora and Ziziphora clinopodioides Lamarck. Pilate glandular trichomes were found in Acanthomintha lanceolata Curran, Acanthomintha obovata Jeps., Dorystaechas hastata Boiss. & Heldr. ex Benth., Salvia canariensis L., Salvia rypara Briq. and Salvia verbenaca L. Capitate and pilate glandular trichomes had a spherical head attached centrically to the stalks (Fig. 4A–F). Salvia dorrii was characterized by capitate glandular trichomes with a pear-shaped head attached excentrically to the stalk (Fig. 1J). Following Cantino (1990), subsessile glands could be divided into several subtypes based on the number of cells and the cell wall configurations in the head of the gland. However, in Mentheae, the number of cells in the head of the gland often varied within the same species, and sometimes the number of head cells could not be counted. Therefore, we focused on the presence or absence of subsessile glands and their density. Glands with four- or eight-celled heads were rather common in Mentheae (Fig. 4G, H). In addition, multicellular head (more than ten cells) glands occurred only in Perovskia Karel. (Fig. 4L). Subsessile glandular trichomes were distributed throughout Mentheae, like capitate glandular trichomes, except in Bystropogon canariensis, Clinopodium vulgare L., Conradina canescens, Hymenocrater bituminosus Fisch. & C.A.Mey., Lallemantia peltata, Lophanthus tschimganicus Lipsky, Prunella vulgaris, Rosmarinus officinalis and Salvia taraxacifolia. LEAF ANATOMY Laminas were dorsiventral or isobilateral (Fig. 5). The epidermis was composed of a single cell layer, and the cells were rectangular or oval on both sides of the leaf. The mesophyll was differentiated into a two-seriate palisade and a one- to four-seriate spongy © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 220 H.-K. MOON ET AL. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE 221 Figure 2. Scanning electron micrographs of leaf surfaces of subtribe Menthinae. A, B, Clinopodium vulgare. Adaxial surface showing irregular epidermal cells. B, Abaxial surface showing distribution of anomocytic/diacytic stomata. C, Cuinia eriantha var. fernandezia. Adaxial surface showing striated isometric epidermal cells. D, Dicerandra christmanii. Adaxial surface showing striated isometric epidermal cells. E, Hedeoma ciliolata. Abaxial surface showing distribution of subsessile glandular trichomes and branched nonglandular trichomes. F, Hoehnea minima. Abaxial surface showing distribution of trichomes. G, H, Mentha pulegium L. G, Adaxial surface showing irregular epidermal cells with diacytic/diallelocytic stomata. H, Abaxial surface showing distribution of trichomes. I, Monarda fistulosa. Abaxial surface showing distribution of trichomes. J, K, Micromeria marginata. J, Adaxial surface showing irregular epidermal cells and striated stomata. K, Abaxial surface showing striated irregular epidermal cells. L, Obtegomeria caerulescens (Benth.) Doroszenko & P.D.Cantino. Abaxial surface of tightly revolute leaf. M, Origanum rotundifolium. Adaxial surface showing isometric epidermal cells with diacytic stomata. N, Piloblephis rigida (W.Bartram ex Benth.) Raf. Abaxial surface of tightly revolute leaf. O, Satureja thymbra. Adaxial surface showing irregular epidermal cells and striated diallelocytic stomata. ! parenchyma. Palisade tissue was present below the upper and lower epidermis (Fig. 5H, J, K), and cells were cylindrical in transverse section. The spongy parenchyma cells, circular or ovoid, were located between the palisade tissues (Fig. 5G, H, J). The midrib region was well developed and projected outwards (Fig. 5A–I). Vascular bundles were collateral. The xylem faced the AD side, and the phloem the AB side. Vascular bundles were covered with parenchymatous cells. Collenchymatous cells occurred below the upper and lower epidermis in the midrib region. In Micromeria marginata, Monardella odoratissima Benth., Saccocalyx satureioides Coss. & Durand and Thymus serphyllum, vascular bundles in the midrib functioned as a mechanical tissue composed of compact thick-walled sclerenchymatous cells (Fig. 5H, I). DISCUSSION LEAF MICROMORPHOLOGICAL CHARACTERISTICS OF MENTHEAE The leaf characteristics of Mentheae show a great diversity in epidermal cell outline, stomatal complex and structure and density of trichomes, and the pattern of epidermal anticlinal cell walls may be closely related to ecological conditions. In general, straight or curved walls are characteristic of species growing in drier conditions, whereas undulate walls are found in species inhabiting more humid areas (Stace, 1965). However, our results do not support this hypothesis. Undulate or sinuate anticlinal walls often appeared in species occurring in xeric habitats, including Hedeoma ciliolata, Poliomintha glabrescens and Ziziphora clinopodioides. In addition, more than one anticlinal wall type was sometimes found within the same leaf (Table 1). Cantino (1990) found five different types of stomata in Mentheae. El-Gazzer & Watson (1968) investigated stomatal configurations in a wide range of Lamiaceae, but listed only the predominant type in each genus. Furthermore, their observations regarding many genera of Lamioideae conflicted with Cantino’s findings (1990). Stomatal position and types from the present study are comparable with those in the study of Cantino (1990). Anomocytic, diacytic and diallelocytic stomatal types were most common in Mentheae, but anisocytic and actinocytic stomata were sometimes found together with anomocytic or diacytic stomata (Table 1). It is notable that all examined taxa had mainly anomocytic or diacytic stomata. Mentheae commonly had amphistomatic leaves, which are believed to occur more frequently in xeric habitats (Parkhurst, 1978). In the amphistomatic species, stomata were more numerous on the AB surface. We found only one incongruent case in Monarda fistulosa L., which was reported as hypostomatic (Cantino, 1990), but was amphistomatic with a few stomata on the AD surface in the present study. Trichome diversity in Lamiaceae may be taxonomically significant at various taxonomic levels (Rudall, 1979, 1980; Abu-Asab & Cantino, 1987; Cantino, 1990; Demissew & Harley, 1992; Ayodele & Olowokudejo, 2006). However, the structure and density of trichomes are diverse, and sometimes too variable for phylogenetic use (Guerin, 2005). Trichomes are one of the most important traits contributing to passive resistance of plants to pathogens, pests and drought (Levin, 1973), and may play an important role in the adaptation to environments with high levels of irradiance (Stenglein et al., 2005). The types of trichome, however, are usually constant in species groups (Stace, 1965; Okpon, 1969). The leaf epidermis of Pistacia atlantica Desf. (Anacardiaceae), for instance, has been shown to have the same trichome type across several populations under different climatic conditions, although the density of trichomes differed among the populations according to the altitude (Belhadj et al., 2007). In Mentheae, the types of trichome recognized were consistent with previously published data (Bokhari & Hedge, 1971; Cantino, 1990; Kaya et al., 2000; Jang & © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 222 H.-K. MOON ET AL. Figure 3. Light and scanning electron micrographs of nonglandular trichomes of tribe Mentheae. A, Blephilia ciliate Raf. Simple short nonglandular trichomes (< 100 mm). B, Micromeria marginata. Short uniseriate nonglandular trichomes (< 200 mm). C, Glechoma hederacea. Structure of simple unicellular nonglandular trichome. D, Conradina grandiflora. Simple long nonglandular trichomes (> 100 mm). E, Clinopodium vulgare. Long uniseriate nonglandular trichomes (> 200 mm). F, Minthostachys mollis (Kunth) Griseb. Structure of long uniseriate nonglandular trichome. G, Conradina canescens. Cross-section of leaf with structure of simple short nonglandular trichomes and short uniseriate nonglandular trichomes. H, Clinopodium vulgare. Distribution of simple short and long uniseriate nonglandular trichomes on the leaf margin. I, Rosmarinus officinalis. Branched nonglandular trichomes at abaxial surface. J, K, Hedeoma ciliolata. J, Branched nonglandular trichomes. K, Structure of branched nonglandular trichome. L, Perovskia abrotanoides. Branched nonglandular trichome. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE 223 Figure 4. Light and scanning electron micrographs of glandular trichomes of tribe Mentheae. A, Clinopodium acinos Kuntze. Short stalked capitate glandular trichome. B, Lepechinia calycina Epling. Capitate glandular trichome. C, Nepeta cataria. Cross-section of leaf; capitate glandular trichome on the abaxial surface with stoma on the adaxial surface. D, Rosmarinus officinalis. Cross-section of leaf showing structure of capitate glandular trichome on the adaxial surface. E, F, Acanthomintha obovata. E, Pilate glandular trichomes on petiole. F, Pilate glandular trichomes on the adaxial surface. G, Nepeta nuda. Glands with four-celled head on the abaxial surface. H, Blephilis ciliata. Glands with eight-celled head on the abaxial surface. I, J, Cross-section of leaf showing structure of subsessile glandular trichome on the abaxial surface. I, Glechoma hederacea. J, Origanum vulgare. K, Nepeta cataria. Cross-section of leaf showing capitate glandular trichome and simple trichome on the midrib on the adaxial surface. L, Perovskia abrotanoides. Multicellular head (more than ten) glands with branched nonglandular trichome on the abaxial surface. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 224 H.-K. MOON ET AL. Figure 5. Light micrographs of cross-sections of leaves in Mentheae. A–J, Midrib structure with vascular bundle. A, Perovskia scrophulariifolia. B, Rosmarinus officinalis. C, Salvia glutinosa. D, Agastache urticifolia. E, Mentha pulegium. F, Prunella vulgaris. G, Cunila origanoides, showing xylem fibres and phloem fibres. H, I, Vascular bundle modified into a mechanical tissue. H, Micromeria marginata. I, Thymus serphyllum. J, Dicerandra christmanii showing a vascular bundle with an extension area towards the abaxial surface. K, L, Glechoma hederacea. K, Leaf structure with longitudinal section of vein. L, Stomata on the abaxial surface. Hong, 2007; Satil, Ünal & Hopa, 2007). To address the distribution patterns of each trichome type, we used the most comprehensive phylogeny of Mentheae (Walker & Sytsma, 2007; Fig. 6). Non-glandular trichomes, both simple-unicellular and uniseriate, occurred widely in Mentheae. The presence of both kinds of trichome is a common condition. The presence of only uniseriate or unicellular trichomes may represent a diagnostic characteristic. The presence of branched trichomes in most Chloanthoideae Briq. was suggested as a possible synapomorphy for this former subfamily, although branched nonglandular © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE Non-glandular trichome type unicellular uniseriate branched Glandular trichome type capitate pilate subsessile Salvia aethiopis Salvia sclarea Salvia canariensis Salvia taraxacifolia Salvia officinalis Perovskia abrotanoides* Rosmarinus officinalis Salvia polystachya Salvia rypara Salvia dorrii Meriandra bengalensis Dorystaechas hastata Zhumeria majdae Salvia glutinosa 225 Salviinae Lepechinia caulescens* Lepechinia calycina* Melissa officinalis Clionopodium chinensis* Clionopodium vulgaris* Conradina canescens Conradina grandiflora* Cunila origanoides* Poliomintha longiflora* Poliomintha incana* Hedeoma ciliolata* Hoehnea epilobioides Pycnanthemum albescens* Pycnanthemum incanum* Pogogyne douglasii* Glechon marifolia Pogogyne serpylloides* Poliomintha glabrescens* Acanthomintha obovata* Monarda fistulosa Monarda punctata* Dicerandra christmanii* Dicerandra odaratissima Rhododon ciliatus Menthinae I Ziziphora capitata* Ziziphora clinopodioides* Satureja thymbra* Satureja acinos* Mentha pulegium* Thymus pallasianus* Thymus serpyllum Origanum vulgare Origanum rotundifolium* Glechoma hederacea Schizonepeta multifida Agastache urticifolia Nepeta cataria Drepanocaryum sewerzowskii Nepetinae Lycopus uniflorus Cleonia lusitanica Prunella vulgaris Horminum pyrenaicum Menthinae II Ocimum basilicum Ocimeae (outgroup) Figure 6. Diagrams of trichome types in Mentheae and their distribution on the most recent molecular phylogenetic tree (simplified tree based on Walker & Sytsma, 2007, Figs 3, 4). For each taxon the occurrence of stomata on the adaxial side of leaves is shown within the clades: Black, amphistomatic leaves, white, hypostomatic leaves. The taxa indicated by an asterisk were included in the present study but lacking in Walker & Sytsma (2007), and their phylogenetic position is estimated by assuming that the genera are monophyletic. trichomes were also found in Ocimeae Dumort. and Lamioideae (Cantino, 1990). However, our results show that the presence of branched trichomes evolved at least twice, even within Mentheae (Fig. 6). Branched trichomes occurred mainly in Salviinae, but this type of trichome was also found in Hedeoma ciliolata and Neoeplingia leucophylloides of subtribe Menthinae (Table 1). Glandular trichomes were almost universally present in Mentheae (Fig. 6); their absence is a characteristic of only Conradina canescens and Prunella vulgaris. Capitate glandular trichomes were observed in all taxa of Salviinae and were also present in Mentheae. The absence of capitate glandular trichomes is a possible synapomorphy for certain species. Subsessile glandular trichomes have been widely reported in Lamiaceae (Solereder, 1908; Metcalfe & Chalk, 1950; Huang & Cheng, 1971; Bosabalidis & Tsekos, 1982; Werker et al., 1985; Cantino, 1990). Our results also show a wide distribution of subsessile glandular trichomes in Mentheae (Fig. 6). Subsessile glandular trichomes with multicellular © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 226 H.-K. MOON ET AL. heads were restricted to Perovskia (Fig. 4L), whereas four- to eight-celled head subsessile glands were rather common throughout the tribe (Fig. 4G, H). Glandular trichomes normally co-occurred with nonglandular trichomes, but, in Cuminia eriantha var. fernandezia (Colla) Harley, Cunila origanoides (L.) Britton, Hoehnea minima, Lophanthus tschimganicus, Origanum rotundifolium Boiss., Pogogyne douglasii Benth. and Thymus serpyllum, glandular trichomes were observed alone. The subtribal delimitation of Mentheae has been questioned by molecular studies (Wagstaff et al., 1995; Walker & Sytsma, 2007). Although all published molecular phylogenetic analyses are largely congruent with the subtribal delimitation of Mentheae sensu Harley et al. (2004), the monophyly of Menthinae and Nepetinae is still questioned. Because the phylogenetic significance of pollen and nutlet features, including sexine ornamentation, morphology of abscission scar and surface pattern of nutlets, was demonstrated (Moon, 2008; Moon et al., 2008a, b), we might expect that additional morphological characteristics could help to solve the remaining taxonomic problems in Mentheae, especially at the subtribal level. Although the leaf morphological characteristics are too variable to be used phylogenetically, it is too early to draw conclusions on the systematic importance of leaf morphology in Mentheae. Indeed, a thorough molecular phylogeny of Mentheae is still lacking. Our results confirm that leaf morphological characteristic are useful for the identification of species in Mentheae. LEAF MICROSTRUCTURE AND FLORAL DIMORPHISM The present study included the gynodioecious Glechoma hederacea L. (separate hermaphroditic and female plants coexist in a single population) and the distylic Perovskia abrotanoides (two types of flower with different style lengths). Gynodioecy is rather common in Lamiaceae, whereas heterostyly has been reported in only a few species (Owens & UberaJiménez, 1992; Moon et al., 2008b). Floral size dimorphism between two flower types within gynodioecy (hermaphrodite + female) and heterostyly (pin + thrum) is well known in flowering plants (Delph, 1996). Significant size dimorphism between different floral morphs has been found in Glechoma hederacea (Widén, 1992) and Perovskia abrotanoides Kar. (H.-K. Moon, unpubl. data). However, we could not find micromorphological differences between different flower morphs. This is consistent with the former study of a gynodioecious species Lycopus maackianus Makino (Hong & Moon, 2003). Indeed, the leaf micromorphological features were almost identical, except for the density of certain trichome types (Table 1). This result might suggest that the micromorphological characteristics of leaves are more closely associated with species circumscription than with gender dimorphism. LEAF ANATOMICAL STRUCTURE The midrib vasculature and anatomical structure of leaves were generally uniform throughout the tribe. In Micromeria marginata, Monardella odoratissima, Saccocalyx satureioides and Thymus serpyllum, the vascular bundle of the midrib was modified into a mechanical tissue. This is often found in plants which grow in seasonally dry or nutrient-poor environments (Rudall, 1980). Micromeria marginata, Saccocalyx satureioides and Thymus serpyllum are tiny shrubs and have small leaves with revolute margins. These are all typical xeromorphic adaptations (Rudall, 1979, 1980). The presence of glandular trichomes is another xeromorphic characteristic (Rudall, 1980), although glandular trichomes occur throughout the tribe (Table 1). Further leaf anatomical studies of Mentheae with expanded taxon sampling will be necessary to ascertain whether the variation in leaf microstructure reflects phylogenetic relationships among species, or whether it may be influenced significantly by ecological factors. CONCLUSIONS The systematic value of leaf epidermal micromorphological characteristics was restricted to the subtribal level in Mentheae. Branched nonglandular trichomes were found throughout subtribe Salviinae, but it was not an apomorphic trait. The great diversity of stomata and trichomes in Mentheae was useful at rather lower taxonomic levels (genus or species). Subsessile glandular trichomes with multicellular heads are diagnostic for the genus Perovskia. Salvia dorrii is easily recognized by trichomes with pear-shaped heads attached excentrically to the stalks. Rosmarinus officinalis is characterized by a few capitate glandular trichomes at the AD surface with a cluster of branched nonglandular trichomes at the AB surface. Therefore, the combination of leaf micromorphological characteristics could be helpful in the identification of species. Nonetheless, the value of these features might be better appreciated in Mentheae by means of a phylogenetic approach when used in conjunction with other morphological and molecular characteristics. ACKNOWLEDGEMENTS We thank the directors of the herbaria BR, G, GH, K, LV, MO, S and SNU for permission to examine speci- © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE mens, either through loans or during visits. Sincere thanks are also due to the editor, Michael Fay, and two anonymous reviewers for their insightful comments on the manuscript, and to Hassan Rankou for editorial help. We are also grateful to Anja Vandeperre and Nathalie Geerts (K.U. Leuven) for technical assistance. The Fund for Scientific Research-Flanders (FWO, G.0268.04 and G.0250.05) and K.U. Leuven (OT/05/35) financially supported this research. H.-K. Moon is a postdoctoral fellow at K.U. Leuven (PDMK/ 08/085). REFERENCES Abu-Asab MS, Cantino PD. 1987. Phylogenetic implications of leaf anatomy in subtribe Melittidinae (Labiatae) and related taxa. Journal of the Arnold Arboretum 68: 1–34. Amelunxen F. 1964. Elektronenmikroskopische Untersuchungen an den Drüsenschuppen von Mentha piperita L. Planta Medica 12: 121–139. Amelunxen F. 1965. Elektronenmikroskopische Untersuchungen an den Drüsenschuppen von Mentha piperita L. Planta Medica 13: 457–473. Amelunxen F, Wahlig T, Arbeiter H. 1969. Über den Nachweis des ätherischen Öls in isolierten Drüsenhaaren und Drüsenschuppen von Mentha piperita L. Zeitschrift für Pflanzenphysiologie 61: 68–72. APG II. 2003. 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Carnoy: a new digital measurement tool for palynology. Grana 41: 124–126. Solereder H. 1908. Systematic anatomy of the dicotyledons. Oxford: Clarendon Press. Stace CA. 1965. Cuticular studies as an aid to plant taxonomy. Bulletin of the British Museum (Natural History) Botany 4: 1–78. Stace CA. 1984. The taxonomic importance of the leaf surface. In: Heywood VH, Moore DM, eds. Current concepts in plant taxonomy, Vol. 25. London: Academic Press, 67–93. Stenglein SA, Arambarri AM, Menendez-Sevillano MC, Balatti PA. 2005. Leaf epidermal characters related with plant’s passive resistance to pathogens vary among accessions of wild beans Phaseolus vulgaris var. aborigineus (Leguminosae-Phaseoleae). Flora 200: 285–295. Stevens PF. 2001. Angiosperm phylogeny website. Version 6. May 2005. Available at http://www.mobot.org/MOBOT/ research/APweb/ Wagstaff SJ, Olmstead RG, Cantino PD. 1995. Parsimony analysis of cpDNA restriction site variation in subfamily Nepetoideae (Labiatae). American Journal of Botany 82: 886–892. Walker JB, Sytsma KJ. 2007. Staminal evolution in the genus Salvia (Lamiaceae): molecular phylogenetic evidence for multiple origins of the staminal lever. Annals of Botany 100: 375–391. Webster GL, Del-Arco-Aguilar MJ, Smith BA. 1996. Systematic distribution of foliar trichome types in Croton (Euphorbiaceae). Botanical Journal of the Linnean Society 121: 41–57. Werker E, Putievsky E, Raavid U. 1985. The essential oils and glandular hairs in different chemotypes of Origanum vulgare L. Annals of Botany 55: 93–801. Widén M. 1992. Sexual reproduction in a clonal gynodioecious herb Glechoma hederacea. Oikos 63: 430–438. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE 229 APPENDIX VOUCHER SPECIMENS OF TRIBE MENTHEAE Subtribe Salviinae Chaunostoma mecistandrum Donn. Sm. Dorystaechas hastata Boiss. & Heldr. ex Benth. Lepechinia calycina (Benth.) Epling ex Munz Lepechinia caulescens (Ortega) Epling Meriandra bengalensis (Roxb.) Benth. Perovskia abrotanoides Kar. – thrum type Perovskia abrotanoides Kar. – pin type Perovskia scrophulariifolia Bunge Rosmarinus officinalis L. Rosmarinus officinalis L. Salvia aethiopis L. Salvia canariensis L. Salvia coccinea Buc’hoz ex Etl. Salvia dorrii (Kellogg) Abrams Salvia glutinosa L. Salvia officinalis L. Salvia officinalis L. Salvia polystachya Ort. Salvia pratensis L. Salvia rypara Briq. Salvia sclarea L. Salvia taraxacifolia Coss. & Bal. Salvia verbenaca L. Salvia verticillata L. A, A, S A, A, S A, A, S A S A, A, S A, S A A, S A, S A, S S Zhumeria majdae Rech.f. & Wendelbo S Subtribe Menthinae Acanthomintha lanceolata Curran Acanthomintha obovata Jeps. Blephilia ciliata (L.) Benth. Bystropogon canariensis (L.) L’Hér A, S S S A, S Cleonia lusitanica (L.) L. Clinopodium acinos (L.) Kuntze Clinopodium chinense (Benth.) Kuntze Clinopodium vulgare L. Conradina canescens A. Gray Conradina grandiflora Small Cuminia eriantha var. fernandezia (Colla) Harley Cunila origanoides (L.) Britton Cunila origanoides (L.) Britton Cyclotrichium origanifolium (Labill.) Manden. Dicerandra christmanii Huck & Judd Dicerandra odoratissima R.M.Harper Glechon marifolia Benth. Gontscharovia popovii (B.Fedtsch. & Gontsch.) Boriss. Hedeoma ciliolata (Epling & Stewart) Irving Hesperozygis nitida (Benth.) Epling Hoehnea epilobioides (Epling) Epling Hoehnea minima (Schmidt) Epling Horminum pyrenaicum L. Hyssopus officinalis L. Hyssopus officinalis L. A, S S S A, S S S A A, A, S A, A, A, A, A, S A, S A S S S S S S S S S S S S S S S S S S S S S S EXAMINED IN THE PRESENT STUDY Mexico, 06.xi.1939. Matuda 3915, GH Turkey, 05.xi.1998. Verlooue 3102, BR USA, 24.v.1986. Dechamps 4201, BR Mexico, 04.ix.1890. Pringle 3264, BR Yemen, 12.ix.1977. Lavranos & Newton 15796, MO Nepal, 04.vii.1976. Billiet and Leonard 6805, BR Nepal, 11.vii.1976. Billiet and Leonard 6888 BR Cultivated in Gent: 09.vi.2005. Moon, LV Spain, 18.ix.1983. Cnops 83.56, BR Belgium, 09.vi.2005. Moon, LV Hungary, viii.1876. Rechter s.n., BR Spain, 04.x.1995. Hanson GC95-62, BR Spain, 25.xii.1973. Lewalle 7322, BR USA, 23.v.1965. Cronquist 10171, BR France, 02.ix.1985. Dechamps 2810, BR Spain, 1915. Elías 2450, LV Cultivated in BR: 22.vi.2005. Moon, LV Mexico, 03.x.1980. Rodriguez 5019, BR France, 1973. Witte 17388, LV Bolivia, 07.xi.1993. Billiet & Jadin 6123, BR France, 24.vii.1953. Andre 11, BR Morocco, 09.vii.1984. Lewalle 11087, BR Without locality, 1919 Sennen s.n., LV The Netherlands, 14.vii.1949. Bakhuizen & Van den Brink 6794, BR Iran, 08.iv.1975. Wendelbo & Foroughi 15731, K USA, 05.v.1919 Mason 12284, BR USA, 18.v.1919. Walker 5094, BR USA, 13.vi.1966 Radford 44758, BR France, without date, Bullemont 1855 (hermaphrodite), BR Morocco, 13.v.1934. Wall 45, S Cultivated in KEW 1989-3009: 04.vii.2006. Moon, LV South Korea, without date1931 Mori s.n., SNU Cultivated in KEW 1994-2824: 04.vii.2006. Moon, LV USA, 30.xo.1969. Godfrey 69283, BR USA, 24.x.1956. Ahles & Bell 21395, BR Chile, 23.xi.1991. Billiet & Jadin 5631, BR USA, 07.ix.1897 Anonymous 323b, BR USA, 09.vi.1972. Kral 48356, K Lebanon, 06.vii.1897. Bornmüller 1260, BR USA, 11.ix.1987. Skean, Jr. 2130, MO Turkey, 17.ix.1967. Radford & Leonard 11479, BR Uruguay, 12.ii.1955. Pedersen 3627, BR Russia, 31.viii.1931. Anonymous s.n., K Mexico, 01.x.1954. Rzedowski 5003, GH Brazil, 22.ix.1976. Dombroswski 6442, K Brazil, 23.x.1974. Kummrow 688, K Brazil, 26.i.1916. Dusén 17542, GH Italy, 26.vi.1969. Cnops 21169, BR Ecuador, 22.ix.1974. Bondía et al., 1242GF, BR Cultivated in Kew 1975-1170: 03.vii.2006. Moon, LV © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 230 H.-K. MOON ET AL. APPENDIX Continued Kurzamra pulchella (Clos) Kuntze Lycopus europaeums L. Mentha pulegium L. Micromeria marginata (Sm.) Chater Minthostachys mollis Griseb Minthostachys andina (Britton ex Rusby) Epling Monarda fistulosa L. Monarda punctata L. Monardella macrantha A.Gray Monardella nana A.Gray Monardella odoratissima Benth. Neoplingia leucophylloides Ramamoorthy Obtegomeria caerulescens (Benth.) Doroszenko Origanum rotundifolium Boiss. Origanum vulgare L. Origanum vulgare L. Pentapleura subulifera Hand.-Mazz. Piloblephis rigida (Bartram ex Benth.) Raf. Pogogyne douglasii Benth. Pogogyne serpylloides (Torr.) A.Gray Poliomintha glabrescens A.Gray ex Hemsl. Poliomintha incana (Torr.) A.Gray Poliomintha longiflora A.Gray Prunella vulgaris L. Pycnanthemum albescnes Torr. & A.Gray. Pycnanthemum incanum (L.) Michx. Rhabdocaulon coccineum (Benth.) Epling Rhabdocaulon strictum (Benth.) Epling Rhododon ciliatus (Benth.) Epling Saccocalyx satureioides Coss. & Durand Satureja thymbra L. Stachydeoma graveolens (Chapm. ex A.Gray) Small Thymbra spicata L. Thymbra spicata L. Thymus pallasianus Heinr.-Braun Thymus serpyllum L. Zataria multiflora Boiss. Ziziphora capitata L. Ziziphora clinopodioides Lam. S A A, A, S A, S S S S S A, A, S S A A, A, S A, S S S A, S A, A, S A, A, A, S S A S A, S S A, Subtribe Nepetinae Agastache urticifolia (Benth.) Kuntze Cedronella canariensis (L.) Webb & Berthel Dracocephalum parviflorum Nutt. Dracocephalum ruyschiana L. Drepanocaryum sewerzowii (Regel) Pojark. Glechoma hederacea L. – hermaphrodite Glechoma hederacea L. – female Hymenocrater bituminosus Fisch. & C.A.Mey. Lallemantia peltata (L.) Fisch. & C.A.Mey. Lallemantia royleana (Benth.) Benth. Lophanthus tschimganicus Lipsky Marmoritis rotundifolia Benth. Meehania urticifolia (Miq.) Makino Nepeta cataria L. Nepeta cataria L. Nepeta grandiflora L. A, A, A, S A, A, S S A, A, S S A, S A A, S S S S S S S S S S S S S S S S S S S S S S S S S Chile, i.1924. Werdermann 263, GH Belgium, 07.viii.2005. Moon, LV Cultivated in Kew 1994-1897: 04.vii.2006. Moon, LV Cultivated in Kew 1995-1960: 04.vii.2006. Moon, LV Ecuador, 10.viii.1939. Asplund s.n., BR Bolivia, 22.viii.1993. Audivio 585, BR USA, 06.viii.1973. Bouharmont 8498, BR USA, 26.x.1957. Ahles & Haesloop 38096, BR Cultivated in Kew 1980-998: 03.vii.2006. Moon, LV Cultivated in Kew 1999-270: 03.vii.2006. Moon, LV USA, 29.viii.1969. Howell 46064, BR Mexico, 05.viii.1982. Medrano 12792, K USA, 16.viii.1986. Cuadrov & Gentry 2706, MO Cultivated in Kew 1968-19106: 04.vii.2006. Moon, LV France, 1971. Witte 17047, LV Belgium, 22.vi.2005. Moon, LV Iraq, 4–9.vii.1957. Rechinger 12085, K USA, 13.ii.1995. Holst et al. 4543, MO USA, 31.v.1892. Bioletti, BR USA, 28.iv.1964. Rose 64044, BR Mexico, 18.viii.1937. Wynd 696, GH USA 18.vi.1985. Whiting 756/731, GH Mexico, 11.ix.1955. Rzedwskiz 6583, GH Belgium, 17.vi.2005. Moon, LV USA, 27.viii.1982 Kessler et al. 2648, BR USA, 10.ix.1966. Bradley et al. 3491, BR Brazil, 12.iv.1977. Harley 20332, K Argentina, 18.iv.1979. Persen 12455, GH USA 06.vi.1969. Correll 37399, GH Algeria, 26.v.1965. Faurel et al. 5650, BR Cultivated in Kew 2001-823: 04.vii.2006. Moon, LV USA, 18.viii.1962. Godfrey 62494, BR France, 18.vi.1883. Ascherson 470, BR Cultivated in Kew 2001-825: 03.vii.2006. Moon, LV Cultivated in Kew 2001-4194: 04.vii.2006. Moon, LV Cultivated in Kew 1973-21043: 04.vii.2006. Moon, LV Iran, 16.v.1892. Bornmüller 4274, GH Moldova, 03.vi.1971. Diaconescu s.n., BR Turkey, 01.ix.1993, Vašák s.n., BR USA, 26.vi.1996. Bouharmont 26820, BR Spain, Canary Is., 29.vi.1926. Linder 2670, GH Canada, 22.vi.1978. Collet 111, BR Russia, 28.vii.1981. Vašák s.n., BR Tajikistan, 23.v.1974. Vašák and Ziatník s.n., BR Belgium, 19.v.2005. Moon, LV Belgium, 19.v.2005. Moon, LV Iran, 28.vi.1942. Kyilbynacob s.n., BR Without locality, vi.2002. Dagh s.n., BR Iran, 04.v.1972. Léonard 5385, BR Uzbekistan, 09.viii.1926. Baranov & Raikova s.n., BR China, 20.vii.1979. Hartmann 2464, G Japan, viii.1936. Makino s.n., BR France, 18.vii.1975. Kapp s.n., LV Cultivated in Kew 1994-1793: 04.vii.2006. Moon, LV Cultivated in BR: 22.vi.2005. Moon, LV © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231 LEAF MICROMORPHOLOGY AND ANATOMY OF MENTHEAE 231 APPENDIX Continued Nepeta nuda L. Nepeta fissa C.A.Mey. Schizonepeta multifida (L.) Briq. S S A, S Schizonepeta tenuifolia (Benth.) Briq. A, S Switzerland, Valais, 06.vii.1973. Lawalree 18115, LV Armenia, 04.vii.1894. Sintenis 6096, BR Russia, 20.vii.1974. Amebyehko and ChnpnIIehko s.n., MO Japan, without date, Makino s.n. 1932, BR Unplaced genera Heterolamium debile (Hemsl.) C.Y.Wu Melissa flava Benth. Melissa officinalis L. Melissa officinalis L. A, S S S A China, A. Henry s.n., March, 1889, K Nepal, without date, Kumaon et al. 1, BR France, 1986. Sotiaux s.n., BR Cultivated in Kew 1994-2690: 03.vii.2006. Moon, LV A, anatomical observation; S, scanning electron microscopy observation. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 211–231